Innate immunity is the first line of defense against microbial invasions in the human body. Innate immune cells recognize invading microorganisms via a set of receptors termed Toll-like receptors (TLRs). Activation of TLRs triggers rapid innate immune responses and subsequently leads to effective adaptive immune responses to combat the foreign invaders. The innate immune responses are mediated by expression of a variety of immune response genes and synthesis of inflammatory mediators such as cytokines, lipid secondary metabolites, and reactive oxygen species. Dysfunctional responses, either failure to respond or hyperresponsiveness, may lead to both acute and chronic immunodeficiency and inflammatory diseases. Therefore, the intensity and duration of TLR activation must be tightly controlled. We have identified a novel TLR interacting protein termed TriadSA which is a member of a group of related proteins resulting from alternative splicing of the Triads gene. The TriadSA is an E3 ubiquitin-protein ligase that promotes ubiquitination and degradation of several TLRs and their downstream signaling molecules. Overexpression of TriadSA blocks TLR ligand-induced NF-kappaB activation in cells. Conversely, reduction in endogenous TriadSA by siRNA increases TLR expression and enhances TLR activation. These results suggest that TriadSA is a component of an endogenous control mechanism for TLR expression and activation. In this project, we will focus on three specific aims to gain more insight into its functions. We will: (1) perform structure-functional studies to determine the binding motif within the TLRs for TriadSA, (2) determine the functions and molecular mechanisms of TriadSA in TLR ligand-induced cellular responses, and (3) determine the roles of TriadSA in LPS induced tolerance and septic shock. Completion of this project will help us to elucidate the control mechanisms of TLR activation, and provide information for potential therapeutic strategies in diseases caused by loss of control of TLR activation.